High-bionic in-vivo immune microenvironment in-vitro skin model and application

Abstract

The invention discloses a high-bionic in-vivo immune microenvironment in-vitro skin model and application, and belongs to the technical field of biological medicine. The preparation method of the in-vitro skin model of the high-bionic in-vivo immune microenvironment comprises the following steps: firstly, adding a culture medium into the lower layer of a six-pore plate, then adding a mouse bone marrow cell suspension, then putting the six-pore plate into a Transwell chamber, flatly laying separated mouse skin on an absorptive gelatin sponge, putting the absorptive gelatin sponge into the Transwell chamber, adding the culture medium into the Transwell chamber, and then putting the mouse skin into the transwell chamber to obtain the in-vitro skin model of the high-bionic in-vivo immune microenvironment. Placing the six-hole plate in a cell incubator for culturing to obtain the in-vitro skin model. According to the in-vitro skin-bone marrow cell co-culture system, an immune environment is provided for the in-vitro skin by introducing bone marrow cells, so that the culture environment of the in-vitro skin is more complete, the difficulties that cell components are simple and the immune environment is deficient at present can be solved, and the in-vitro skin-bone marrow cell co-culture system is more in line with the 3R principle and is high in bionic property and low in cost.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an ex vivo skin model of a highly biomimetic in vivo immune microenvironment and its application. Background Technology

[0002] Skin is the largest organ in the human body and contains abundant immune cells, such as Langerhans cells and T cells, which play a crucial role in infection, allergies, autoimmune diseases, and tumor immune surveillance. The application of ex vivo skin culture technology is essential for studying changes in skin under different physiological and pathological states and exploring drug interventions on the skin. Currently, the main ex vivo skin models used are as follows, each with certain limitations: (I) Monolayer skin cell culture, such as keratinocyte and fibroblast culture. This model uses only one type of cell and cannot simulate the complex skin tissue environment; (II) Co-culture of epidermal keratinocytes and fibroblasts using separate chambers. Although this model upgrades the 2D culture system to a 3D culture system, it cannot simulate the dermal environment due to the homogeneity of a single cell. Under these 3D conditions, epidermal cells are in only one state, making it impossible to distinguish epidermal keratinocytes from different layers such as the spinous and granular layers, and it lacks immune cells, making it impossible to study the role of immune cells; (III) Simple ex vivo skin models. [1-5] In these models, cultured human skin exhibits varying degrees of cell death within 24 hours of ex vivo culture and fails to simulate genuine skin-immune cell interactions. In summary, current ex vivo skin culture techniques all have significant shortcomings, and in vitro skin simulation technology requires further refinement. Therefore, establishing a highly biomimetic, low-cost ex vivo skin-immune cell co-culture system is of great significance for skin immunology research, drug screening and efficacy evaluation, and personalized medicine.

[0003] In addition, due to the difficulty in obtaining human ex vivo skin, using animal (primarily mouse) ex vivo skin for alternative experiments is more common and widely accepted by researchers. Compared to directly using mice as experimental subjects, using mouse skin ex vivo culture models better conforms to the "Replacement" and "Reduction" principles of the 3R principle, and can significantly reduce the cost of experiments or drug screening.

[0004] According to a Data Bridge Market Research report, the global dermatology drug market is projected to reach $73.5 billion by 2032, with a CAGR of 10.25% from 2025 to 2032. The psoriasis drug market, one of the immune-inflammatory skin diseases, is expected to dominate the dermatology segment with a 51.15% market share in 2025. Therefore, the development of reliable preclinical research models will effectively improve the success rate of drug development, providing a platform for pharmaceutical companies to conduct more precise drug screening and for patients to receive more precise treatment.

[0005] Bone marrow cells contain various mature or immature immune cells, such as monocytes, neutrophils, natural killer cells, B cells, and T cells. [6] The isolation procedure for mouse bone marrow cells is simple and the technology is mature. It is widely used in basic research. Among them, bone marrow-derived macrophages (BMDM) formed by the adhesion of bone marrow cells stimulated by macrophage colony-stimulating factor (M-CSF) are the most widely used. However, a single immune cell cannot accurately simulate the immune microenvironment that the skin comes into contact with. Moreover, the development of skin diseases is often the result of the combined action of multiple immune cells, which brings challenges to the research of immune inflammatory skin diseases.

[0006] Based on this, the present invention designs a highly biomimetic, low-cost ex vivo skin-bone marrow cell co-culture system using bone marrow cells to provide an immune environment for ex vivo skin, in order to prepare an ex vivo skin model with a biomimetic in vivo immune microenvironment.

[0007] References:

[0008] [1] EVANS EA, SAYERS SR, KODJI X, et al. Psoriatic skin inflammation induces a pre-diabetic phenotype via the endocrine actions of skin secretome[J]. Mol Metab, 2020, 41:101047.

[0009] [2]MURAMATSU T,TADA H,KOBAYASHI N,et al.Induction of the 72-kD heatshock protein in organ-cultured normal human skin[J].J Invest Dermatol,1992,98(5):786-90.

[0010] [3]COMPANJEN AR,VAN DER WEL LI,WEI L,et al.A modified ex vivo skinorgan culture system for functional studies[J].Arch Dermatol Res,2001,293(4):184-90.

[0011] [4]SHANNON JL,KIRCHNER SJ,ZHANG J Y.Human Skin Explant Preparationand Culture[J].Bio Protoc,2022,12(18).

[0012] [5] Tianjin University of Science and Technology. An in vitro simulated skin model: China, CN106568911A[P]. 2017-04-19.

[0013] [6]BACCIN C, AL-SABAH J, VELTEN L, et al.Combined single-cell and spatial transcriptomics reveal the molecular, cellular and spatial bone marrowniche organization[J]. Nat Cell Biol, 2020, 22(1):38-48. Summary of the Invention

[0014] One objective of this invention is to provide an ex vivo skin model that is highly biomimetic to the in vivo immune microenvironment, which is prepared using the following steps:

[0015] Step 1: Isolate mouse bone marrow cells and resuspend them in culture medium;

[0016] Step 2: Separate the mouse skin and set aside.

[0017] Step 3: First, add culture medium to the lower layer of the six-well plate, then add the mouse bone marrow cell suspension from Step 1, and then place it in a Transwell chamber. Lay the mouse skin from Step 2 flat on an absorbent gelatin sponge and place it in the Transwell chamber. Add culture medium to the Transwell chamber and place the six-well plate in a cell culture incubator to obtain the ex vivo skin model.

[0018] Furthermore, the culture medium is EMEM medium.

[0019] Furthermore, in step 3, the volume of the lower layer culture medium in the six-well plate is 1-1.5 mL, and the bone marrow cells are added at a concentration of 1-10 × 10⁶ cells / mL. 6 Add medium at a density of / mL to the lower layer of a six-well plate; add 0.5mL of medium to the Transwell chamber.

[0020] Furthermore, the culture conditions in step 3 are 37°C and 5% CO2 for 48 hours.

[0021] To further expand the application scope of the ex vivo skin model of this invention, modeling and intervention operations can also be performed on ex vivo skin or bone marrow cells. For skin pretreatment, gene editing technologies such as CRISPR / Cas9 or RNA interference can be used to edit specific mouse skin cells to obtain ex vivo skin with specific genes knocked out, knocked down, or overexpressed. Alternatively, specific compounds can be applied topically to the surface of the ex vivo skin to simulate skin damage or skin diseases for pathological studies of skin damage or diseases, as well as studies related to the skin's immune microenvironment. Specific compounds or drugs can be applied to the skin surface or added to the culture medium to simulate transdermal or systemic drug administration routes for drug screening and efficacy evaluation studies, or studies on the effects of drugs on the local immune microenvironment. For bone marrow cell pretreatment, gene editing technologies such as CRISPR / Cas9 or RNA interference can be used to knock out, knock down, or overexpress specific genes in bone marrow cells. Overexpression and co-culture of treated cells with ex vivo skin can be used to explore the functional effects of specific genes on specific bone marrow cells in a specific skin microenvironment or to explore personalized treatment strategies for skin diseases in individuals with abnormal expression of specific immune cell genes. Bone marrow cells can be pretreated with compounds or drugs for a specific time before being co-cultured with ex vivo skin. This method is suitable for evaluating the efficacy of drugs on target immune cells or the overall immune response induced by specific treatments in bone marrow cells within a simulated specific skin physiological or pathological microenvironment, and for drug screening and efficacy evaluation. The duration of co-culture can also be dynamically controlled according to experimental objectives, such as studying the early events and mid-to-late-stage progression mechanisms of skin inflammation, and assessing the impact of different drug intervention time points on pharmacological mechanisms and pharmacodynamics.

[0022] A second objective of this invention is to provide applications for the aforementioned highly biomimetic in vivo immune microenvironment-based ex vivo skin model, including applications in dermatopathology research, skin immunology research, drug screening, and efficacy evaluation. This application is for non-disease diagnostic purposes.

[0023] This invention introduces bone marrow cells to provide an immune environment for ex vivo skin, making the culture of ex vivo skin more complete. It can solve the current difficulties of simple cell components and lack of immune environment, and is more in line with the 3R principle. It is a highly biomimetic and low-cost ex vivo skin-bone marrow cell co-culture system. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the ex vivo skin culture model of the present invention.

[0025] Figure 2 HE staining results of mouse skin tissue cultured on gelatin sponge for 48 hours.

[0026] Figure 3 HE staining results of mouse skin tissue cultured on filter paper for 48 hours.

[0027] Figure 4 In the middle, the left side shows the HE pathological staining results of skin tissue co-cultured in different bone marrow cell cultures for 48 hours, and the right side shows the survival rate of cells in the lower chamber detected by flow cytometry staining FVS780 under the corresponding conditions.

[0028] Figure 5 In the middle, the left side shows the HE pathological staining results of skin tissue co-cultured in different bone marrow cell cultures for 48 hours, and the right side shows the survival rate of cells in the lower chamber detected by flow cytometry staining FVS780 under the corresponding conditions.

[0029] Figure 6 HE staining results of the model after single UVB irradiation followed by dexamethasone treatment to improve UVB-induced acute skin damage. D, E, and F are magnified views of A, B, and C, respectively.

[0030] Figure 7 for Figure 6 Results of flow cytometry analysis of the proportion of pro-inflammatory (CD86-positive) macrophages in the corresponding groups. Detailed Implementation

[0031] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0032] The reagents and materials used in the following examples include: 0.4μm pore size, 24mm diameter Transwell chambers, absorbent gelatin sponges (Jiangxi Xiang'en Medical Technology Development Co., Ltd., specifications 20*20*5mm, standard type 6PCS), DMDM ​​culture medium (containing 10% fetal bovine serum, Gibco). TM Antibiotics (antifungal agents), erythrocyte lysis buffer, EMEM medium (containing 15% newborn calf serum, Gibco) TMAntibiotics / antifungal agents), Epilife medium (containing bovine pituitary extract, recombinant human insulin-like growth factor-I, hydrocortisone, bovine transferrin, epidermal growth factor, etc.), monocyte magnetic bead sorting kit (Miltenyi Biotec, Cat. No: 130-100-629, Monocyte Isolation Kit (BM), mouse), FcR blocker (BDPharmingen, 553142), APC-CY7 FVS780 dye (BD Pharmingen, 565388), PerCP-cy5.5F4 / 80 (Thermo Fisher Scientific, 45-4801-80), PE-CY7 CD86 (BD Pharmingen, 560582), 0.75% dexamethasone (dexamethasone raw material dissolved in anhydrous DMSO, concentration 0.75%, 20 μL of solution applied to each skin patch).

[0033] Example 1

[0034] 1. Skin isolation from mice: The backs of C57 mice were shaved, and the mice were euthanized. The mice were then immersed in 75% alcohol for about 5 minutes. Under aseptic conditions, the shaved skin from the backs of the mice was cut off using sterile ophthalmic scissors and forceps, laid flat on a 10cm cell culture dish, and divided into small pieces of skin approximately 8mm*15mm for later use.

[0035] 2. Isolation of mouse bone marrow cells: Under aseptic conditions, the femur and tibia of mice were isolated, and muscle and fibrous tissue were removed from the bones. The ends of the bones were cut off with sterile scissors to expose the bone marrow cavity. 5 mL of DMDM ​​culture medium (containing 10% fetal bovine serum, Gibco) was drawn using a 5 mL syringe. TM (Antibiotics and antifungal agents) Using sterile ophthalmic forceps, hold the bone upright above a 6cm cell culture dish. Insert a syringe needle into the bone marrow cavity to flush out the bone marrow. Repeat the flushing process twice until the bone turns white. Collect the cell suspension from the 6cm cell culture dish into a 15mL centrifuge tube. Filter the cell suspension through a 70μm cell sieve into a new 15mL centrifuge tube. Centrifuge at 300g for 10 minutes, discard the supernatant, add red blood cell lysis buffer, mix well, and let stand for about 10 minutes. Centrifuge at 300g for 10 minutes, discard the supernatant, add an appropriate amount of culture medium to resuspend the cells, and count them for later use.

[0036] 3. Model Building

[0037] During preliminary experiments, the inventors discovered that the ex vivo skin culture conditions reported in existing literature did not yield good skin survival rates, and were even less able to simulate the inflammatory microenvironment of the skin in vitro. Therefore, they designed and constructed an ex vivo skin model capable of simulating the in vivo immune microenvironment. Since ex vivo skin culture requires support and the maintenance of the gas-liquid interface, simply using filter paper or gelatin sponges is insufficient. Thus, Transwell chambers were introduced. Transwell chambers ensure a continuous permeation supply of nutrients to the upper ex vivo skin tissue while maintaining the gas-liquid interface, preventing skin edema, ensuring that the lower fluid does not exceed the gas-liquid interface, and providing a feasible platform for the interaction between immune cells and ex vivo skin within specific chambers.

[0038] In this invention, the constructed model culture system is as follows: first, culture medium is added to a six-well plate, then bone marrow cell suspension is added, and then the plate is placed in a Transwell chamber. The excised skin is laid flat on an absorbent gelatin sponge and placed in the Transwell chamber. Culture medium is added to the Transwell chamber and the plate is then placed in a cell culture incubator for culture.

[0039] 3.1 Survival analysis of isolated mouse skin

[0040] First, the results of culturing mouse excised skin on gelatin sponges were examined. The specific experimental groups were as follows: (1) Mouse excised skin was spread on gelatin sponges and then placed directly on the bottom of a six-well plate. 1.5 mL of EMEM medium was added, but bone marrow cells were not added, and the plate was cultured for 48 h; (2) 1.5 mL of EMEM medium was added to the six-well plate, and then bone marrow cells were cultured at a ratio of 1×10⁻⁶. 6 Add / mL density to a six-well plate, then gently place the gelatin sponge carrying the excised skin on the surface of the liquid and incubate for 48h; (3) First add 1.5mL of EMEM medium to the lower layer of the six-well plate, then add bone marrow cells at a density of 1×10 6 Add / mL density to the bottom layer of a six-well plate, then place it in a Transwell chamber, add 0.5mL of EMEM medium in the Transwell chamber, and then gently place the gelatin sponge carrying the excised skin on the surface of the liquid in the chamber and incubate for 48h; (4) Replace the EMEM medium in group (1) with Epilife medium, and keep the other treatment conditions the same; (5) Replace the EMEM medium in group (2) with Epilife medium, and keep the other treatment conditions the same; (6) Replace the EMEM medium in group (3) with Epilife medium, and keep the other treatment conditions the same.

[0041] HE staining was performed on the isolated skin obtained from the above experiments. The HE staining procedure was as follows: the isolated skin was carefully removed with forceps and laid flat on a filter paper of appropriate size, fixed in 4% paraformaldehyde, then embedded in paraffin and sectioned. The white slides were dewaxed: xylene 10 min - xylene 10 min - anhydrous ethanol 5 min - anhydrous ethanol 5 min - 95% ethanol 5 min - 85% ethanol 5 min - 70% ethanol 5 min - pure water 5 min; staining treatment: hematoxylin 4 min - rinse with tap water 10 min - hydrochloric acid ethanol differentiation 3 seconds - rinse with tap water 10 min (blue return) - eosin 25 seconds - 1 min; dehydration treatment: anhydrous ethanol 5 min - anhydrous ethanol 5 min - anhydrous ethanol 5 min; permeabilization treatment: xylene 10 min - xylene 10 min - xylene 10 min; mounting treatment: after mounting with neutral resin, the slides were allowed to air dry naturally, observed under a microscope and images were acquired.

[0042] like Figure 2 As shown, the skin in A, B, and C was more intact than that in D, E, and F. The dermis and epidermis of the skin in D, E, and F were severely separated, almost completely separated, indicating that Epilife medium could not maintain the survival of mouse ex vivo skin on gelatin sponges. Although the dermis and epidermis of the skin in A and B were not completely separated, there was partial separation. In C, not only were bone marrow cells added, but also absorbable gelatin sponges with the ex vivo skin laid flat were placed on the chamber, which improved the dermis and epidermis separation in A and B.

[0043] Next, the results of culturing mouse excised skin on filter paper were examined. The specific experimental groups were as follows: (1) The excised skin was laid flat on filter paper and then placed directly on the bottom of a six-well plate. 1.5 mL of EMEM medium was added, but bone marrow cells were not added. The plate was cultured for 48 h; (2) 1.5 mL of EMEM medium was added to the six-well plate first, and then bone marrow cells were added at a ratio of 1×10⁻⁶. 6 Add / mL density to a six-well plate, then gently place the filter paper containing the excised skin on the surface of the liquid and incubate for 48h; (3) First add 1.5mL of EMEM medium to the six-well plate, then add bone marrow cells at a density of 1×10 6 Add / mL to a six-well plate, drill several holes in the filter paper with a syringe needle, and then gently place the filter paper containing the excised skin on the surface of the liquid and incubate for 48h; (4) Replace the EMEM medium in group (1) with Epilife medium, and keep the other treatment conditions the same; (5) Replace the EMEM medium in group (2) with Epilife medium, and keep the other treatment conditions the same; (6) Replace the EMEM medium in group (3) with Epilife medium, and keep the other treatment conditions the same.

[0044] HE pathological staining was performed on the isolated skin obtained from the above experiments, following the same procedure as above.

[0045] like Figure 3 As shown, the skin in A, B, and C was more intact than the skin in D, E, and F. The dermis and epidermis of the skin in D, E, and F were severely separated, almost completely separated, indicating that Epilife medium could not maintain the survival of mouse skin excised on transfer paper. Although the dermis and epidermis of the skin in A, B, and C were not completely separated, partial separation was present. Even though bone marrow cells were added to C and the excised skin was placed on perforated transfer paper, the dermis and epidermis separation in A and B was not improved.

[0046] Based on the above results, mouse skin samples need to be laid flat on absorbent gelatin sponges, placed in a small chamber, and kept alive in EMEM medium.

[0047] 3.2 Survival analysis of bone marrow cells used for co-culture

[0048] Next, the survival of bone marrow cells used for co-culture was examined. The specific experiment was divided into 3 groups: (1) First, 1.5 mL of EMEM medium was added to the lower layer of a six-well plate, and then bone marrow cells were cultured at a ratio of 1×10⁻⁶. 6 Add / mL density to the lower layer of a six-well plate, then place it in a Transwell chamber, add 0.5mL of EMEM medium to the Transwell chamber, and then gently place the gelatin sponge carrying the excised skin on the surface of the liquid in the chamber and incubate for 48h; (2) first take 1×10 6 / mL bone marrow cell resuspension 1mL sorted mononuclear cells according to the magnetic bead sorting kit steps, and resuspended them with 1mL DMEM medium, added to the lower layer of a six-well plate, and then placed in a Transwell chamber. 0.5mL DMEM medium was added to the Transwell chamber, and then the gelatin sponge carrying the excised skin was gently placed on the liquid surface in the chamber and cultured for 48h; (3) First take 1×10 6 1 mL of bone marrow cell resuspension was used to sort monocytes according to the magnetic bead sorting kit steps, and the cells were resuspended in 1 mL of EMEM medium. The cells were then added to the bottom layer of a six-well plate and placed in a Transwell chamber. 0.5 mL of EMEM medium was added to the Transwell chamber, and a gelatin sponge carrying excised skin was gently placed on the surface of the liquid in the chamber and cultured for 48 h.

[0049] HE pathological staining was performed on the isolated skin obtained from the above experiments, following the same procedure as above.

[0050] The viability of cells in the lower layer of the Transwell chamber was then detected by flow cytometry staining with FVS780. The procedure for flow cytometry cell viability detection was as follows: cells were collected at 300g and centrifuged for 10 minutes at 10⁻⁶ ppm. 6Taking a single cell as an example, resuspend the cells in 100 μL of PBS containing FcR blocker, incubate for 20 minutes, then add APC-CY7 FVS780 dye, PerCP-cy5.5 F4 / 80, and PE-CY7 CD86. Incubate at 4°C for 20 minutes in the dark, centrifuge at 300g for 10 minutes, discard the supernatant, resuspend in PBS, centrifuge again, discard the supernatant, resuspend in 300 μL of PBS, and perform flow cytometry analysis. FlowJo software gating strategy: First, use FSC-A and SSC-A to gating the target cells, then use FSC-A and FSC-H to gating non-adhesive cells, then use APC-CY7 negative cells to gating live cells, then use PerCP-cy5.5 positive cells (macrophages) to gating the live cells, and further gating PE-CY7 positive cells (pro-inflammatory macrophages).

[0051] like Figure 4 As shown, when the lower layer of the chamber contains mononuclear cells, regardless of whether the lower layer culture medium is EMEM or DMEM, the skin shows partial separation of the dermis and epidermis. When the culture medium is DMEM, the epidermal cells even exhibit obvious nuclear shrinkage, indicating the death of the epidermal cells. Furthermore, the flow cytometry results show that the survival rate of mononuclear cells is low. Only when the lower layer culture medium of the chamber is EMEM and the co-cultured cells are bone marrow cells, no obvious separation of the dermis and epidermis is observed, and the survival rate of bone marrow cells is high.

[0052] The effects of co-culturing skin tissue with different bone marrow cell cultures were further investigated. Specifically, the culture was divided into two groups: (1) 1.5 mL of EMEM culture medium was added to the lower layer of a six-well plate, and then bone marrow cells were cultured at a ratio of 1×10⁻⁶. 6 Add / mL density to the lower layer of a six-well plate, then place it in a Transwell chamber, add 0.5mL of EMEM medium to the Transwell chamber, and then gently place the gelatin sponge carrying the excised skin on the surface of the liquid in the chamber and incubate for 48h; (2) first take 1×10 6 1 mL of bone marrow cell resuspension was used to sort monocytes according to the magnetic bead sorting kit. The cells were resuspended in 1 mL of monocyte-specific culture medium and added to a six-well plate. The plate was then placed in a Transwell chamber. 0.5 mL of monocyte culture medium was added to the Transwell chamber. A gelatin sponge carrying excised skin was then gently placed on the surface of the liquid in the chamber and cultured for 48 hours.

[0053] The isolated skin obtained from the above experiments was subjected to HE pathological staining, and the cell viability was detected using the same method as above.

[0054] like Figure 5As shown, when the lower layer of the chamber is a mononuclear cell-specific culture medium, the HE results of isolated mouse skin showed no obvious dermal-epidermal separation and no obvious epidermal cell nuclear shrinkage morphology, but the flow cytometry results showed that the survival rate of mononuclear cells was still low.

[0055] Based on the above results, it can be seen that when the lower layer culture medium in the chamber is EMEM and the co-cultured cells are bone marrow cells, the skin condition is better, with no obvious dermal-epidermal separation or cell nuclear shrinkage, and the survival rate of bone marrow cells is higher.

[0056] Therefore, the model construction method of the present invention is as follows: isolated mouse bone marrow cells are processed at a ratio of 1-2 × 10⁻⁶. 6 Disperse the sample at a density of 1-1.5 mL in 1-1.5 mL of EMEM medium and add it to the bottom layer of a six-well plate. Then add the Transwell chambers. Spread the mouse skin on an absorbent gelatin sponge and place it in the Transwell chamber. Add 0.5 mL of EMEM medium to the Transwell chamber and incubate at 37°C in a 5% CO2 cell culture incubator for 48 hours.

[0057] Example 2

[0058] Application of ex vivo skin models in drug screening

[0059] Sunburn, a common skin disease in clinical practice, is an acute inflammatory response of the skin after excessive exposure to UVB radiation, manifested as erythema, edema, blisters, and desquamation. In basic and applied research, a single UVB irradiation of the back skin of mice is often used to simulate sunburn. Dexamethasone is a commonly used drug in clinical treatment of sunburn, and in this example, it is used as a positive control to evaluate the application value of this model in screening new drugs for the treatment of UVB-targeted skin damage.

[0060] The experiment was divided into three groups: (1) First, 1.5 mL of EMEM culture medium was added to the lower layer of a six-well plate, and then bone marrow cells were added at a ratio of 1×10⁻⁶. 6 / mL density was added to the lower layer of a six-well plate, and then placed into a Transwell chamber. 0.5 mL of LEMEM medium was added to the Transwell chamber, and then the gelatin sponge carrying the excised skin was gently placed on the surface of the liquid in the chamber. After culturing for 48 h, it was the untreated group (Sham group); (2) the excised skin was subjected to 430 mJ / cm 2 After a single UVB irradiation, the skin was spread flat on a gelatin sponge and cultured according to the conditions of the untreated group, which was the UVB group; (3) 20 μL of 0.75% dexamethasone solution was evenly applied to the surface of the isolated skin with a pipette tip, and left to stand for a while until absorbed. Then, the isolated skin was irradiated according to the UVB group and cultured according to the conditions of the untreated group, which was the UVB+dexamethasone group.

[0061] like Figure 6 As shown, after a single UVB irradiation of isolated mouse skin, the nuclei of epidermal cells showed varying degrees of shrinkage, breakage, and even disappearance, indicating epidermal cell death. Dexamethasone alleviated this UVB-induced acute isolated skin damage.

[0062] Figure 7 for Figure 6 The proportion of pro-inflammatory (CD86-positive) macrophages was determined by flow cytometry for each group. Following a single UVB irradiation of mouse skin, the F4 / 80 ratio was observed in co-cultured bone marrow cells. + CD86 in macrophages + The proportion of cells was significantly increased, and dexamethasone significantly inhibited the proportion of pro-inflammatory macrophages in co-cultured bone marrow cells, suggesting a good alleviating effect on UV-induced inflammation.

[0063] The results above show that the in vitro model constructed in this invention can simulate common skin injuries and can be used to evaluate the screening of intervention drugs for skin injuries and the effect of drugs on the immune microenvironment of skin immune damage.

Claims

1. A highly biomimetic ex vivo skin model of an in vivo immune microenvironment, characterized in that, Prepared using the following steps: Step 1: Isolate mouse bone marrow cells and resuspend them in culture medium; Step 2: Separate the mouse skin and set aside. Step 3: First, add culture medium to the lower layer of the six-well plate, then add the mouse bone marrow cell suspension from Step 1, and then place it in a Transwell chamber. Lay the mouse skin from Step 2 flat on an absorbent gelatin sponge and place it in the Transwell chamber. Add culture medium to the Transwell chamber and place the six-well plate in a cell culture incubator to obtain the ex vivo skin model.

2. The ex vivo skin model according to claim 1, characterized in that, The culture medium is EMEM medium.

3. The ex vivo skin model according to claim 1, characterized in that, In step 3, the volume of the lower layer culture medium in the six-well plate is 1-1.5 mL, and the bone marrow cells are added at a rate of 1-10 × 10⁶ cells / well. 6 Add medium at a density of / mL to the lower layer of a six-well plate; add 0.5mL of medium to the Transwell chamber.

4. The ex vivo skin model according to claim 1, characterized in that, The culture conditions in step 3 are 37℃ and 5% CO2 for 48 hours.

5. The application of the ex vivo skin model of the highly biomimetic in vivo immune microenvironment as described in any one of claims 1-4, characterized in that, The applications include dermatopathology research, dermatoimmunology research, drug screening, and efficacy evaluation.

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